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. 2008 Jun 24;5(6):e137. doi: 10.1371/journal.pmed.0050137

Figure 9. Transcriptional Responses to Renal IP and Expression of the A2BAR within the Kidneys.

Figure 9

(A–D) Age-, gender-, and weight-matched C57BL/6J mice were subjected to right nephrectomy followed by in situ IP with a hanging weight system for atraumatic occlusion of the left renal artery. The IP protocol consisted of four cycles of ischemia/reperfusion (4 min each), followed by indicated times of reperfusion. Transcriptional responses of the A1AR, A2AAR, and A2BAR of the A3AR were assessed by real-time RT-PCR. Data were calculated relative to β-actin and are expressed as fold change compared to sham-operated animals without IP treatment.

(E) Western blotting for renal A2BAR following IP treatment. Kidneys were excised at indicated times, flash frozen, and lysed; proteins were resolved by SDS PAGE and transferred to nitrocellulose. Membranes were probed with anti-A2BAR antibody. The same blot was reprobed for β-actin as a control for protein loading. One representative experiment of three is shown.

(F–I) A previously described A2BAR reporter mouse was used (A2BAR-KO/β-gal–knock-in mice). Renal tissues were prepared and stained for β-galactosidase (β-Gal) as indicative of the A2BAR gene promoter, which drives the expression of this reporter gene. Procedures are as detailed under Methods. (F, G) β-Gal staining of kidney derived from littermate controls (WT) or A2BAR−/− mice, and captured at the indicated magnifications (16 ×) with an Olympus IX70 microscope combined with Hamamatsu charge-coupled device camera C4742–95.

(H, I) HE-staining histological examination of renal sections derived from the kidney shown in upper panel. Arrows point to β-Gal stained blood vessels. Fold magnification of captured images are 600×. WT mice were subjected to similar staining showing no expression. β-Gal expression in kidney is clear and localized only in blood vessels.

Statistics: (A–D) One-way ANOVA with Dunn's multiple comparison test (**, p < 0.001 versus control WT mice without IP [C]). The F-test results are (A) F (3,17) = 19.82, p < 0.001.